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The carbon magnetic resonance spectra of a series of C-19 hydroxy, C-5β,6β epoxy, C-5α,6α epoxy, and C-24 ethyl steroids have been assigned. Shift parameters for the C-19 hydroxyl function are listed. The data presented has led to a reassignment of the published spectra of several C5α,6 α epoxy steroids. In addition, 13Cmr spectral data for several bicyclic enones and a Δ5(10)-3-estrone are presented.

The steroid analogue 4,4a,5,6,7,8-hexahydro-2(3H)-naphthalenone was hydroxylated at C-8α, C-8β, and C-4a by Rhizopusarrhizus. Similar products were obtained by peracid oxidation of the corresponding enol ethers: hydroxylation of estr-4-ene-3,17-dione by the same fungus occurred at the analogous C-6 and C-10 positions. These results are consistent with a mechanism of microbial hydroxylation involving the enol form of the Δ4-3-ketone. Data from the incubations with R. arrhizus of androst-4-ene-3,17-dione specifically labelled with deuterium at C-4, C-6α, or C-6β and from those of other deuterium labelled substrates have been interpreted in terms of a mechanism of C-β hydroxylation involving a rate-determining step before enolization of the ketone, followed by rapid enolization and oxidation of the enol to give the 6β-hydroxy-Δ4-3-ketone. The kinetic isotope effect, kH/kD, for the hydroxylation of androst-4-ene-3,17-dione at C-6β has been found to be 1.2 ± 0.1.

The products of the incubation of androst-5-ene-3,17-dione (2a), 3β-hydroxyandrost-5-ene-17-one (2b), and androsta-3,5-diene-17-one (3) with Rhizopus arrhizus ATCC 11145 under a variety of conditions have been identified and the mechanisms of their formation discussed. In addition, several C-(4,5)- and C-(5,6)-epoxyandrostanes have been incubated with R. arrhizus, the products identified and possible pathways for their formation presented.

The products arising from the incubations of C-6α and C-6β chloro-, fluoro-, and methyl-substituted Δ4-3-keto steroids with the C-6β hydroxylating fungus Rhizopusarrhizus ATCC 11145 have been identified, and their formation rationalised in terms of Δ3,5 enolic intermediates. The incubations of C-6 chloro and methyl Δ3,5 enol acetates with R. arrhizus, and the oxidations by m-chloroperoxybenzoic acid of the corresponding enol ethyl ethers have also been described. The relevance of the results so obtained to the mechanism of C-6β hydroxylation of Δ4-3-keto steroids by R. arrhizus is discussed.